A supervised heatmap of 11 developmentally-regulated myeloid genes.

<p>Using the expression data from the Affymetirix Mouse Exon 1.0ST arrays, we created a supervised heatmap with SPOTFIRE using z-score averaging of the probeset with the highest average expression for each of 11 developmentally-regulated myeloid genes across all of our flow-sorted bone marrow cell samples. The legend is shown below the heatmap with downregulated genes in green and upregulated genes in red.</p

A supervised heatmap of the 22 genes significantly dysregulated in both the CMP and GMP compartments.

<p>We created a supervised clustering using SPOTFIRE of the 22 genes that were significantly dysregulated (unadjusted p value<0.001, fold change ≥2) in both the CMP and GMP ANOVA results comparing WT vs. <i>Ctsg-PML-RARA</i> samples. The supervised heatmap was created by plotting the relative expression of each of these genes to each other as a relative percentage, rather than z-score averaging, since the differences in expression levels between some genes was large, and z-score averaging inappropriately highlighted the genes with the highest expression levels. The legend is shown below the heatmap, with minimally expressed genes in green, and highly expressed genes in red.</p

Expression of genes used to target <i>PML-RARA</i> expression in mice.

<p>Expression data for the indicated genes in flow-sorted bone marrow cells using murine Affymetrix Exon 1.0ST arrays or Human Genome U133 Plus 2.0 arrays. Each panel is a highly representative probe on the array. A. <i>cFes</i> (probeset 4758608). B. <i>Itgam</i> (CD11b, probeset 4782002). C. <i>S100A8</i> (<i>MRP8</i>, probeset 202917). D. <i>S100a8</i> (<i>Mrp8</i>, probeset 5010279). E. <i>PML</i> (probeset 235508). F. <i>Pml</i> (probeset 4885841). We have previously published the expression profile of <i>PML</i> in AML and human hematopoietic cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046529#pone.0046529-Welch1" target="_blank">[11]</a>. Panel E includes an additional 86 AML cases of AML and data from a second representative probe.</p

Expression of <i>Ctsg</i> in flow-sorted bone marrow cells and mouse leukemia samples.

<p>Expression profile in indicated WT and <i>Ctsg-PML-RARA</i> (labeled mCG-PR) flow-sorted bone marrow cells and 15 <i>Ctsg-PML-RARA</i> leukemia samples (labeled Mouse APL) using Nugen amplified mRNA and Affymetrix Mouse Exon 1.0ST arrays. We plotted <i>Ctsg</i> expression for probeset 5542324. Note the absence of <i>Ctsg</i> mRNA in <i>Ctsg</i> deficient cells (labeled Ctsg KO). There were no statistically significant differences in <i>Ctsg</i> expression between the same cell populations, when comparing WT and <i>Ctsg-PML-RARA</i> samples (using two-tailed t test, p-value cutoff 0.05). Comparing the levels of Ctsg expression between the <i>Ctsg-PML-RARA</i> samples using a two-tailed t test, the p-value for SLAM vs. KLS = 0.11, KLS vs. CMPs = 0.00026, CMPs vs. GMPs = 0.022, GMPs vs. MEPs 2.4×10⁻⁸, MEPs vs. Pros 2.6×10⁻⁷, GMPs vs. Pros = 0.027, Pros vs. PMNs = 2.4×10⁻⁶.</p

Effects of <i>Ctsg-PML-RARA</i> on multi-lineage hematopoiesis.

<p>A. Effects on myeloid and lymphoid lineage hematopoiesis. Bone marrow cells from indicated mice at 6 weeks of age were mixed at ratios of 1∶1, 9∶1, or 1∶9 with competitor CD45.1⁺/CD45.2⁺ bone marrow cells from sex- and age-matched mice. These cells were transplanted into sex-matched, 6-week-old, lethally irradiated CD45.1⁺ recipients. At the indicated time points, peripheral blood was assessed for ratios of CD45.2⁺ and CD45.1⁺/CD45.2⁺ white blood cells within the B220⁺, CD3⁺, or Gr1⁺ compartments. One sample, two-tailed t-test compared outcomes with the expected values of 10%, 50%, or 90%. Alpha was set at 0.05. Time points with p<0.01 (*) and p<0.05 (†) are indicated. B–C. Effect of <i>Ctsg-PML-RARA</i> on erythroid lineage hematopoiesis. Bone marrow cells from 6-week-old and 8-week-old, healthy mice were plated in methylcellulose containing erythropoietin. B. Total colonies after one week in culture (each data point represents results from an individual mouse in three combined experiments). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046529#s2" target="_blank">Results</a> of paired t-test between WT and mCG-PR samples are shown (p-value = 0.002). C. After one week in culture, total colony cells were washed and the number of immature erythrocytes (cKit⁺CD71^dim) was assessed. Results of a paired t-test between WT and mCG-PR samples are shown (p-value = 0.007).</p

Sequencing of iPSC clones.

<p><b>A.</b> Number of variants identified per iPSC clone. <b>B.</b> Variant allele frequencies of all validated mutations for each clone. Samples are ranked by the number of variants in decreasing order. No mutations were identified in Ax2-11, thus it is not listed in panel B.</p

Comparison of hematopoietic potential of iPSCs to mouse ESCs.

<p>Fractions of Lin⁻ cells (A), KLS cells (B), Kit+Lin-Sca-1- Progenitors (C), GMPs (D), CMPs (E), and MEPs (F) from iPSCs relative to mouse ESCs after 7 days of OP9 coculture (unsorted).</p

Generation of iPSC clones from a single mouse C57BL/6 male mouse.

<p><b>A.</b> Schematic of experimental approach (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120585#sec002" target="_blank">Material and Methods</a> for full protocol). <b>B.</b> Representative bright-field images (left) and alkaline-phosphastase stains (right) of B6/BLU ESCs (top) and a representative iPSC (bottom, Ax1-10). All images at 100x magnification. <b>C.</b> Images from the teratoma derived from Ax1-10 demonstrating ectoderm (neural tissue), mesoderm (cartilage) and endoderm (ciliated respiratory epithelium).</p

Validation array coverage.

<p>Validation array coverage.</p

Hematopoietic differentiation potential of the 24 iPSC clones.

<p>100,000 cells from OP9 cocultured mESCs (B6/BLU) or iPSCs were plated in methylcellulose media containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). <b>A.</b> CFUs were counted after 7 additional days of culture. The relative number of CFUs per 100,000 cells plated from Day7 iPSC-derived progenitors vs. Day7 ESC (B6/BLU)-derived progenitors are shown. iPSC clones are ranked from the highest to the lowest average of two independent experiments. Error bars represent the means +/− one standard deviation. <b>B.</b> Morphology of day 7 OP9 cocultured ESC-derived cells after 7–8 days of additional culture in MethoCult media containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). A scale bar of 20 μm is shown. <b>(C-E)</b>. Fractions of CD11b⁺ (<b>C</b>), CD34⁺Kit⁺ (<b>D</b>), and Ter119⁺ (<b>E</b>) cells obtained after 7 days of methylcellulose culture containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo), comparing iPSC-derived progenitors relative to ESC-derived progenitors, in the same order as panel A. <b>F.</b> Lack of correlation between the number of mutations and the hematopoietic differentiation potential of the iPSC clones (r² = 0.0006065).</p